1. Field of the Invention
In one aspect, this invention relates to the establishment and characterization of a schwannoma cell line from a patient with neurofibromatosis 2. In another aspect this invention relates to methods of screening therapeutic treatments for neurofibromatosis 2.
2. Description of the Related Art
Neurofibromatosis 2 (NF2) is a genetic disorder characterized by the formation of multiple benign nervous system tumors, including schwannomas, meningiomas and ependymomas (Martuza, R. L. & tk tk Eldridge, R. 1988 N Engl J Med 318:684-688; Mulvihill, J. J. et al. 1990 Ann Intern Med 113:39-52; Evans, D. G. et al. 1992 Q J Med 84:603-618). These tumors are often benign with slow growth, but their location predominantly within the central nerve system (CNS) may have catastrophic effects on sensitive intracranial and intraspinal structures, thus, causing a high rate of morbidity and mortality. Affected individuals generally develop symptoms of eighth nerve dysfunction, including deafness (often bilateral) and balance disorder (Martuza, R. L. & Eldridge, R. 1988 N Engl J Med 318:684-688; Kanter, W. R. et al. 1980 Neurology 30:851-859). NF2 occurs with an incidence of approximately one in 40,000 live births where about 50% of cases reveal no family history and are likely due to new mutations (Evans, D. G. et al. 1992 J Med Genet 29:841-846).
Clinically, there are two subtypes in NF2 patients (Evans, D. G. et al. 1992 J Med Genet 29:841-846; Ruttledge, M. H. et al. 1996 Am J Hum Genet 59:331-342; Parry, D. M. et al. 1996 Am J Hum Genet 59:529-539; MacCollin, M. et al. 1995 Ann Neurol 38:554-555). Patients with the severe (Wishart) subtype usually have onset by 20 years of age, develop many CNS tumors in addition to bilateral vestibular schwannomas, and exhibit rapid clinical progression that may lead to death by the 3rd or 4th decade (Evans, D. G. et al. 1992 J Med Genet 29:847-852; Parry, D. M. et al. 1994 Am J Med Genet 52:450-461). On the other hand, patients with the mild (Gardner) subtype often do not develop symptoms until the 3rd decade, and have few tumors other than bilateral vestibular schwannomas and have a relatively benign clinical course (Evans, D. G. et al. 1992 J Med Genet 29:841-846; Parry, D. M. et al. 1994 Am J Med Genet 52:450-461).
The NF2 gene is located on chromosome 22q12 (Rouleau, G. A. et al. 1987 Nature 329:246-248). The genetic evidence from NF2 patients with schwannomas (Bijisma, E. K. et al. 1992 Genes Chromosomes Cancer 5:201-205), meningiomas (Dumanski, J. P. et al. 1987 PNAS USA 84:9275-9279), sporadic unilateral vestibular schwannomas (Parry, D. M. et al. 1994 Am J Med Genet 52:450-461) and mutated alleles, together with the loss of heterozygosity (LOH) for restriction fragment length polymorphism (RFLPs) on chromosome 22q, has suggested that the NF2 gene is a tumor suppressor gene.
Genotype-phenotype correlation analysis revealed that more severe disease (Wishart) tends to harbor NF2 mutations that result in premature protein termination due to frameshift mutations (deletions or insertions) or the presence of premature termination codon. In the patients with milder clinical disease (Gardner), the mutations are either missense mutations or due to not being found by ordinary mutation detection methods. It has been predicted that nonsense NF2 mutations could result in unstable truncated proteins whereas missense mutations might generate full-length proteins incapable of growth suppression (Gutmann, D. H. et al. 1998 Hum Mol Genet 7:335-345). In a recent report, differing growth rate were noted in culture when different sporadic vestibular schwannoma tumor tissues were cultured (Pelton, P. D. et al. 1998 Oncogene 17:2195-2209). Unfortunately, no NF2 gene mutation information was provided.
Currently, several biochemical experiments have also indicated that the NF2 protein directly interacts with plasma membrane molecules such as CD44, EBP50 or hNHE-RF/EBP50 and cytoskeleton molecules such as β II-spectrin (Sainio, M. et al. 1997 J Cell Sci 110:224-940). These findings suggest that the NF2 gene product may, like its homologous proteins ezrin, radixin and moesin (ERM), work as a linker between cell membrane and cytoskeleton. This linkage may block cytoplasmic growth signaling from the cell surface. However, a detailed pathway of how NF2 protein mediates the tumor suppression function is still unclear.
The establishment of an NF2-in vitro model is essential to elucidate the NF2 gene tumor 15 suppression function in Schwann cells and as the first step to test new therapeutic approaches. To date, no single cell line has been developed from NF2 tumor cells, and most studies were conducted either in yeast, mouse schwannoma cells or other non-Schwann human cells. There are three main reasons for this limited progress. First, human Schwann cells are difficult to obtain. Second, because of the lack of knowledge of Schwann cell growth factors, once the Schwann cells are obtained, they do not proliferate in culture. Third, there is the contamination of human fibroblast. Recently, we have developed a method for establishing short-term primary schwannoma cells in culture. These primary cultures can be enriched to greater than 99% pure and could be very useful for the study of genetic alterations and NF2 gene functions at the cellular and molecular level (Hung, G. et al. 1999 Int J Oncology 14:409-15).